The training and research plans of this K99/R00 proposal will directly advance the candidate's long-term career goal of developing an interdisciplinary research program that examines epigenetic, genetic and environmental risk factors associated with the development of alcohol use disorders (AUDs). Emerging evidence from animal research indicates that variability and changes in DNA methylation represent a core component of the vulnerability to AUDs. However, the extant research literature is small, and epigenetic studies of AUDs in human subjects are characterized by several limitations: most studies have examined DNA methylation at a identify DNA methylation markers that are reliably associated with behavioral indices of risk for alcohol use disorders (AUDs) in two high-risk samples of males and females Our preliminary analyses, using DNA from buccal cells, provide strong initial support for associations between chronic heavy drinking and methylation at numerous CpG sites in biological pathways relevant to alcohol use. We will seek to replicate and extend this blood mononuclear cells (PBMCs) and buccal cells will be assayed on a customized methylation platform that includes the top (up to 384) CpG sites advanced from our preliminary work. Using a discordant MZ twin design is a powerful strategy for identifying DNA methylation markers associated with alcohol use because it permits an assessment of the epignome independent of genomic sequence variation and other confounding factors. We will also examine the stability of DNA methylation patterns associated with alcohol use across a 3-year period in an independent sample of young adults between 18-21 years, a developmental period in which some individuals are at high risk for making the transition from initial alcohol us to risky drinking behavior. It is anticipated that this work will identify a set of reliable and biologically-relevant CpG markers that may besingle gene, at a single time, and in male-only samples. The overarching goal of the present application is to . work in a monozygotic (MZ) twin sample discordant for chronic heavy drinking. DNA obtained from peripheral prioritized for further research (e.g., with animal models) that seeks to evaluate their causal significance. Career development during the K99/R00 award period is sought in AUDs and epigenetics. Training in these areas will be accomplished through coursework, workshops, conferences, and lab training, and through extensive mentoring and consulting with senior investigators. Overall, the proposed K99/R00 has both theoretical and applied significance: theoretical significance for the light it may shed on individual differences in DNA methylation associated with alcohol use, and applied significance for our ability to eventually advance new biomarkers and develop novel strategies for alcohol treatment based on a better understanding of the role of DNA methylation in the development of AUDs. This work will combine the applicant's expertise in behavioral genetics and genetic epidemiology with the development of new skills that are critical for success as an independent investigator, and will lay the groundwork for future longitudinal epigenetic research on AUDs.